Optimising methods for the recovery and quantification of di- and tripeptides in soil

Di- and tripeptides are intermediaries in the nitrogen cycle and are likely to have roles in the soil-microbeplant continuum, but they have hitherto been difficult to measure in soils. To lay the base for future studies of oligopeptides in soil, we added 10 known di- and tripeptides with diverse chemical properties to forest and agricultural soils and then recovered the peptides by means of induced diffusive fluxes using microdialysis, a minimally-intrusive soil sampling technique. The concentration of the peptides recovered with the probes was 25-39% (relative recovery) of the concentration in the external solution, and followed the same trend as previously observed for amino acids, with smaller peptides (e.g. Gly-Gly) recovered at a higher rate than larger ones (e.g. Tyr-Phe). After derivatisation with AccQ-Tag (TM), a standard method for amino acids, peptides were analysed by ultra-high-pressure liquid chromatography-triple quadrupole mass spectrometry. Multiple reaction monitoring mass spectrometry was used to quantify specific peptides with a short run time of 15 min and a detection limit of 0.01-0.02 pmol injected (0.005-0.01 pmol mL(-1)) for the different peptides. This methodology allowed successful analysis of all standard di- and tripeptides tested here. We conclude that microdialysis in combination with UHPLC-MS will allow measurement of plant-relevant fluxes of di-and tripeptides in undisturbed soil.